Binding assay device with non-absorbent carrier material

ABSTRACT

A method of producing a binding assay device provides a porous membrane comprising a material enabling capillary movement of a liquid sample from a first area on the membrane on a second area on the membrane. A detection site is disposed on the membrane between the first and second areas in a non-absorbent medium disposed on the membrane between the detection site and the membrane first area is attached by an adhesion with a dry reagent disposed between the medium and the membrane in order to enable mobilization of the reagent by passage of a liquid sample therepast.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a divisional of application Ser. No. 10/033,259filed on Dec. 28, 2001.

BACKGROUND OF THE INVENTION

The present invention is generally related to test devices forperforming binding assays for determining characteristics of a sample,such as a biological liquid. More particularly, the present invention isdirected to assays, which utilize porous carrier materials fortransporting reagents.

The device is in the form of test strips commonly utilized in theanalysis of various types of samples, particularly, biological fluids,because of their convenience and rapid determination of results. Teststrips assays for detecting various clinically significant substancesand biological fluids, such as urine and serum, have been veryadvantageous in assisting the diagnosis and treatment of disease states.

Many binding assay formats are well known in the art and includecompetition, sandwiched and agglutination assays.

A thorough discussion of prior art assay devices is set forth in U.S.Pat. No. 5,770,458. These assays commonly use test strips comprising anabsorbent, porous matrix incorporated with indicator reagents usually ofa colorimatrix type. The sample to be tested is contacted with reagentmatrix and the indicator response is observed after a certain period oftime. Combinations of test strips enable simultaneous test reactions toproceed.

A problem associated with prior art devices, as pointed out in the citedreferences, is their lack of uniform mobilization of reagents formigration through the strip. The cited reference utilizes a complicatedmulti-membrane configuration for introducing reagents, which dependsupon parallel liquid migration of liquid sample through co-joinedmembranes.

The present invention provides for a device and procedure forintroducing a binding reagent into a strip absorbent membrane in abinding assay device.

SUMMARY OF THE INVENTION

A binding assay in accordance with the present invention generallyincludes a porous medium comprising a material enabling capillarymovement of a liquid sample from a first area of the membrane to asecond area of the membrane. A detection site is disposed in themembrane between the first and second areas and a non-absorbent mediumis disposed on the membrane between the detection site and the membranefirst area with the medium being attached to the membrane by anadhesive.

A dry reagent is disposed between the medium and the membrane in orderto enable mobilization of the reagent, by passage of the liquid sample,and entry into the membrane and the liquid sample before the liquidsample reaches the detection site.

Preferably, the dry agent disposed between the adhesive and the membraneare disposed in the form of a stripe, with the stripe, being generallytransverse to a direction of the sample migration. Preferably, thestripe is aligned perpendicular to the sample migration direction.

More particularly, the medium, which may be polyester film MYLAR(® is inthe form of a strip having a width greater than a width of reagentstripe. MYLAR(® is a bi-axially oriented, thermoplasitc film made fromethylene glycol and dimethyl terephthalate (DMT) and manufactured byDuPont Teijin Film (Hopewell, Va., USA). It should be appreciated thatthe medium, while referenced to as MYLAR(® may be any polyester, PET orany other label stock well known for adhesive substitution and thatreference to polyester film MYLAR® includes all such alternativemediums. In addition, the membrane is also preferably in the form of astrip having a width at least as wide as the medium width.

The reagent preferably is particle based in an aqueous buffer solutionand the dry reagent is adhered only by the adhesive coated polyesterfilm MYLAR®. In addition, the dry reagent preferably comprises betweenabout 2% and about 30% w/v sugar with the sugar preferably being acrystalline sugar, such as for example, sucrose or mixtures thereof. Amethod in accordance with the present invention of producing a bindingassay device generally includes the steps of providing a porous membranematerial enabling capillary movement of a liquid sample from a firstarea of membrane to a second area of the membrane.

The method further includes disposing a detection site on the membranebetween the first and second areas and providing a non-absorbent mediumhaving a bottom side with an adhesive disposed on the bottom side.

A particle based (i.e. antibody conjugated to colloidal gold) liquidreagent is disposed on the medium and evaporated to provide a dryreagent on the medium bottom side. The device is completed by adheringthe medium bottom side to the membrane between the first area and thedetection site.

More particularly, the non-porous medium is provided with an adhesivecovering the entire medium bottom side and the solublized reagent isdisposed onto the adhesive. Preferably, the evaporated reagent is in theform of a bead along the non-porous medium and the step of evaporatingthe solublized reagent results in a stripe of dry semi-crystallizedreagent along the medium.

In one embodiment of the present invention, the step of evaporatingsolvent includes evaporating the water solvent and the suspendedparticle reagents includes a concentration of sugar.

Through the use of sugar concentration in amounts between about 2% andabout 30% w/v in the particle based reagent, control of the mobilizationrate of the reagent into the membrane upon passage or the liquid sampletherepast is enabled.

The medium may be a polyester MYLAR® tape and the concentration of sugarin the solublized reagent may also be provided to control the viscosityof the solublized reagent applied in bead form to prevent collapse orseparation of the bead upon movement of the medium and drying of thesolublized reagent.

BRIEF DESCRIPTION OF THE DRAWINGS

The advantages and features of the present invention will be betterunderstood by the following description when considered in conjunctionwith the accompanying drawings in which:

FIG. 1 is a perspective of a binding assay device in accordance with thepresent invention generally showing a porous membrane, a detection sitethereon, a sample pad, and end pad along with a non-porous mediumattached to the membrane between the sample pad and the detection site,which includes a dry reagent in contact with the membrane for enablingmobilization thereof as liquid sample passes thereby; and

FIG. 2 is a perspective view similar to FIG. 1 illustrating a method ofthe present invention.

DETAILED DESCRIPTION

With reference to FIGS. 1 and 2, a binding assay device 10 in accordancewith the present invention, generally includes a porous medium 12 whichmay be in the form of a strip as shown. The membrane may be supported ina holder, not shown, in any conventional manner. The membrane may beformed from any bibulous or fibrous material capable of capillaryaction. Specific examples include thin layer chromatography materials,paper or cellulose, porous synthetic plastics including nitro-celluloseand nylon. A sample pad 14 may be provided for receiving a liquid sample16 and wicking the sample into the membrane 12. As hereinabove noted,the membrane comprises a material enabling capillary movement of theliquid sample 16 from a first area 20 adjacent the sample pad to asecond area 22 adjacent and absorbent end pad 24 which may be formedfrom any suitable absorbent material.

A detection site is formed, or disposed in the membrane 12 in anyconventional manner. The detection site may include, for example but notlimited to, an antibody as found in sandwich assays or a drug—BSAconjugate (drug—protein carrier) as found in competitive inhibitionassays.

A non-absorbent medium, such as, for example, MYLAR® 34 is disposed onthe membrane 12 between the first area 20 and the detection site 30 withthe medium 34 being adhered to the membrane 12 by an adhesive 36. Theadhesive 36, may be any conventional pressure sensitive acrylic basedadhesive and preferably, covers an entire surface of the medium 34,although, such coverage is not required.

A dry reagent 40 is disposed between the medium 34 and the membrane 12,and preferably, disposed onto the adhesive 36, underside the medium 34,in the form of a bead, or stripe having a width W₁ smaller than a widthW₂ of the medium 36. The reagent strip 40 is preferably alignedgenerally normal to a longitudinal axis 44 of the membrane 12 in adirection of the sample migration (indicated by the dashed arrow 46).

The membrane 12 is also preferably in the form of a strip which includesa width W₃ which may be equal or greater than the medium width W₂ bothof which are significantly greater than the reagent stripe width W₁. Therelative widths of the medium 36 membrane 12 and stripe 40 may beadjusted for controlling mobilization of the reagent in the strip 40into the membrane as the liquid sample 16 migrates by wicking actiontherepast as indicated by the arrow 46.

As will be hereinafter discussed in connection with the method inaccordance with the present invention, the reagent stripe 40 maycomprise sugars in a crystalline form in order to enable the stripe tobe laid down on the medium 34 and adhesive 36 in form of a liquid beadthat will not collapse upon minor movement of the membrane 12 and mediumand during drying thereof. The concentration of sugar is adjusted tocontrol the rate of mobilization from the stripe 40 into the mediumwhich enables adjustment of the sensitivity of the test being performedby the device.

The method of the present invention includes the providing of the porousmembrane 12 along with disposing the detection site thereon as well asproviding a non-absorbent medium, such as polyester film MYLAR® having abottom side 50 with the adhesive 36 disposed thereon.

The reagent stripe 40 is formed by disposing a particle based reagent onthe adhesive 36 and evaporating a solvent therefrom, preferably water,to provide the dry reagent strip 40.

A formulation of the particle based reagent may comprise an antibodyconjugated to a colloidal gold particle in the size range, for example,of 30-60 nanometers in a buffered sucrose solution in the 2-30 percentw/v range. A non-crystalline sugar, like fructose, can be added in alower percentage to prevent or minimize cracking of the reagent beadupon drying down on the adhesive substrate.

A sugar concentration in the range of 2% to 30% w/v is provided in orderto enable a liquid bead that will not collapse or separate upon movementof the medium 36 and membrane 12 and during drying thereof. For example,a sugar concentration of about 10% enables a bead having a width ofabout 0.05 inches to be formed due to the viscosity provided by thesugar concentration. This is also dependent on the rate and volume ofreagent application.

In addition, it is preferable that a sugar such as fructose be utilizedthat does not crystallize. Fructose can be mixed with a largerproportion of other sugars such as sucrose may be used to preventexcessive cracking as the reagent bead dries on the substrate.

Following the disposition of the reagent 40 onto the medium 34 andadhesive 36 and drying thereof, the medium 34 may be inverted, asindicated by the arrow 60 and adhered to the medium 12 by the adhesive36. The structure of the present invention will tend to eliminate to theundesired reagent retention seen with conventional conjugate releasematerials, thus increasing the repeatability of lateral flow assays.

Although there has been hereinabove described a binding assay device andmethod in accordance with the present invention for the purpose ofillustrating the manner in which the invention may be used to advantage,it will be appreciated that the invention is not limited thereto.Accordingly, any and all modification, variations or equivalentarrangements which may occur to those skilled in the art should beconsidered to be within the scope as defined in the appended claims.

1-22. (canceled)
 23. A method of producing a binding assay device, said method comprising the steps of: providing a porous membrane comprising a material enabling capillary movement of a liquid from a first area of the membrane to a second area of the membrane; disposing a detection site on the membrane between the first and second areas; providing a non-absorbent medium having a bottom side with an adhesive disposed on the bottom side; disposing a solublized reagent onto the adhesive; evaporating a solvent in the particle based reagent to provide a dry reagent on the adhesive; and adhering the medium bottom side to the membrane between the first area and said detection site.
 24. The method according to claim 23, wherein the non-porous medium is provided with adhesive covering an entire medium bottom side.
 25. The method according to claim 24, wherein the evaporated reagent is disposed as a bead along the non-porous medium and the step of evaporation the solvent results in a stripe of dry reagent along the medium.
 26. The method according to claim 25, wherein the step of evaporating the solvent includes evaporating a water solvent. 